Increased drug and pesticide resistance has rendered many control programs for mosquito-borne diseases useless, resulting in an urgent need for new control strategies. Malaria parasites must develop for up to two weeks in the mosquito, and conceptually, this development can be disrupted by enhancing mosquito innate immunity or by shortening the mosquito's lifespan. The insulin/IGF-1 signaling cascade (ISC) regulates both innate immunity and lifespan in Caenorhabditis elegans and Drosophila melanogaster, and, thus, could be manipulated to reduce vector competence of mosquitoes. Based on our preliminary data, exogenous insulin in the bloodmeal modulates not only lifespan and oxidative stress response in female mosquitoes, but also Plasmodium development. To further elucidate these promising results, we will first determine whether exogenous human insulin and IGF-1 can stimulate the ISC in the midgut and other tissues, such as the fat body, in the mosquito Anopheles stephensi. We will also test the impact of these factors on oxidative stress and NO production, key components of aging, innate immunity, and signaling. Next we will engineer An. stephensi mosquitoes to express active forms of two ISC proteins, Akt and PTEN, in the midgut after a bloodmeal. Because AKT activates the midgut ISC and PTEN has the opposite effect, we will be able to answer three questions: (1) Does the midgut ISC regulate oxidative stress and in turn aging and innate immunity in An. stephensi? (2) Does the midgut ISC affect signaling in other An. stephensi tissues? And (3) How do changes to the ISC affect Plasmodium falciparum development in An. stephensi? Finally, we will examine the impact of physiological levels of exogenous human insulin and IGF-1 on the transgenic mosquitoes described above. Insulin levels in human blood can vary by as much as 10-fold after a meal and during malaria parasite infection. By understanding the effects of this naturally occurring variation in human bloodmeals, we can better predict the efficacy of Akt and PTEN over expression on critical variables (e.g., lifespan and immunity) of vector competence. In summary, our proposed work will test both basic and applied hypotheses regarding the ISC and its impacts on mosquito physiology and vector competence that were conceptualized for model invertebrates and mammals. In addition, our work offers a different approach to the transgenic modification of mosquitoes to limit their vectorial capacity. PUBLIC HEALTH RELEVANCE After being ingested in a bloodmeal, malaria parasites, viruses, and nematodes must develop in mosquitoes for extended periods before being transmitted to humans and reservoir hosts. Our goal is to enhance innate immunity or reduce the lifespan of a model mosquito to prevent completion of pathogen development, so that transmission is reduced or eliminated. [unreadable] [unreadable] [unreadable]